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Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R 2  = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

Lyme borreliosis (LB), caused by bacteria of the Borrelia burgdorferi sensu lato (Borrelia) species, is the most common tick-borne infection in the northern hemisphere. LB diagnostics is based on clinical evaluation of the patient and on laboratory testing, where the main method is the detection of Borrelia specific antibodies in patient samples. There are, however, shortcomings in the current serology based LB diagnostics, especially its inability to differentiate ongoing infection from a previously treated one. Identification of specific biomarkers of diseases is a growing application of metabolomics. One of the main methods of metabolomics is nuclear magnetic resonance (NMR) spectroscopy. In the present study, our aim was to analyze whether Borrelia growth in vitro and infection in vivo in mice causes specific metabolite differences, and whether NMR can be used to detect them. For this purpose, we performed NMR analyses of in vitro culture medium samples, and of serum and urine samples of Borrelia infected and control mice. The results show, that there were significant differences in the concentrations of several amino acids, energy metabolites and aromatic compounds between Borrelia culture and control media, and between infected and control mouse serum and urine samples. For example, the concentration of L-phenylalanine increases in the Borrelia growth medium and in serum of infected mice, whereas the concentrations of allantoin and trigonelline decrease in the urine of infected mice. Therefore, we conclude that Borrelia infection causes measurable metabolome differences in vitro and in Borrelia infected mouse serum and urine samples, and that these can be detected with NMR.

Objectives: Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB., Method: We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation., Results: OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10(-6)). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e(-15)). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein., Conclusions: OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.

Bernese Mountain dogs (BMDs) are prone to develop a familial glomerulonephropathy and a pathogenic role of Borrelia burgdorferi sensu lato in this disease has been suspected. Glomerular disease in many affected dogs is clinically inapparent and proteinuria is found incidentally. In this study, urine protein excretion was evaluated in 122 clinically healthy BMDs and 55 controls. The seroprevalence of B. burgdorferi in BMDs was 57%, compared to 16% in controls. There were no significant differences in the occurrence of positive dipstick results, microalbuminuria, urine protein-to-urine creatinine ratio or abnormal urine protein pattern (determined by sodium dodecyl sulphate agarose gel electrophoresis) between BMDs and controls and BMDs with and without antibodies against B. burgdorferi. It was concluded that antibodies against B. burgdorferi are not associated with proteinuria as an early sign of renal disease in BMDs.

Canine Lyme disease is caused by the spirochete Borrelia burgdorferi after transmission by an Ixodes tick, typically resulting in joint pain, fever and lethargy. Lyme nephritis is a poorly characterized syndrome associated with severe glomerular and tubular renal injury and poor clinical outcome in young to middle-aged dogs positive for exposure to B. burgdorferi. The aims of this study were to identify associations between natural exposure to B. burgdorferi and the presence of microalbuminuria in nonclinical young Labrador and Golden Retrievers and to compare two commonly used serologic tests available to document B. burgdorferi exposure: the Western blot and the commercial point-of-care C6 peptide enzyme-linked immunosorbent assay (ELISA) tests. Microalbuminuria was assessed using a commercial point-of-care ELISA specific for canine albumin. Blood and urine samples from 268 asymptomatic Labrador and Golden Retrievers were included. Of these, 18.7% were positive for B. burgdorferi exposure according to the C6 ELISA; 21.2% were positive for natural exposure to B. burgdorferi and 11.5% for vaccinal antibodies according to the Western blot. The agreement rate was 93% between the two tests (kappa = 0.78, P < 0.0001) for natural exposure. Urine from 6.1% of the dogs was positive for microalbuminuria. There was no association between microalbuminuria and exposure to B. burgdorferi based on results of a Western blot (P = 0.57) or C6 ELISA (P = 0.53). Microalbuminuria is likely not a consequence of B. burgdorferi exposure in young nonclinical Labrador and Golden Retrievers.

Diagnosis of Lyme borreliosis by urine polymerase chain reaction (PCR) has been recognized as having better diagnostic sensitivity in patients with erythema migrans than serological methods. We made serial tests with 192 urine specimens from 70 patients with erythema migrans and 60 urine specimens from 21 patients with acrodermatitis chronica atrophicans to evaluate the course of positive urine PCR after antibiotic treatment. Before treatment, urine samples from patients with erythema migrans showed a positive PCR in 27/34 samples (79%), and those from patients with acrodermatitis chronica atrophicans in 7/11 (63%). The specificity of bands was proven by hybridization with GEN-ETI-KTM-DEIA kit in 40/41 samples. Borrelia DNA in urine decreased gradually within the observation period of one year in both patients with erythema migrans and acrodermatitis chronica atrophicans, and persisted without clinical symptoms in 4/45 patients with erythema migrans (8%) after 12 months. Urine PCR can serve as a diagnostic method in early Lyme borreliosis and also in seropositive patients with unclear clinical symptoms.

Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at--20 degrees C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 x g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.

The Roche MagNA Pure automated nucleic acid extraction system was tested for its ability to extract Borrelia burgdorferi DNA from a diverse set of spiked specimen types including blood, cerebral spinal fluid, synovial fluid, urine and ticks. A method comparison between MagNA Pure automated extraction and manual extraction, using either QIAamp columns or phenol/chloroform extraction, showed equivalent detection sensitivities for all methodologies with all specimen types (except for urine, in which case QIAamp extraction was twofold less sensitive). Eighty positive clinical specimens (as determined by an independent testing method), including 76 synovial fluid, and 4 cerebral spinal fluid specimens, were found to be positive by the MagNA Pure/real-time PCR method of extraction and detection. This data shows that the MagNA Pure system can be used to extract B. burgdorferi DNA from clinical specimens, and when combined with real-time PCR, the result is an extremely sensitive assay with limited hands on time and rapid turn around times.

Aim: To evaluate the diagnostic value of the polymerase chain reaction (PCR) to detect Borrelia burgdorferi DNA in patients with ocular Lyme borreliosis., Methods: Of 256 consecutive uveitis patients six selected individuals with clinical evidence for Lyme borreliosis and 30 patients with non-Lyme uveitis were enrolled. Lyme serology was performed by ELISA and western blotting. Urine samples were examined by an optimised nested polymerase chain reaction (PCR) protocol., Results: Only four of six uveitis patients suspected for Lyme borreliosis were ELISA positive, while all six subjects showed a positive western blot. B burgdorferi PCR was positive in all of these six patients. Whereas two of the 30 controls had a positive Lyme serology, B burgdorferi DNA was not detectable by PCR in any sample from these patients., Conclusions: PCR for the detection of B burgdorferi DNA in urine of uveitis patients is a valuable tool to support the diagnosis of ocular Lyme borreliosis. Moreover, these patients often show a weak humoral immune response which may more sensitively be detected by immunoblotting.

The diagnosis of Lyme disease is difficult because tests that reflect active disease or have reasonable sensitivity and specificity are lacking or not timely. Molecular methods are controversial because of differences in assays, gene targets, and limited clinical validation. This review summarizes published assays for Lyme disease diagnosis using skin, plasma, synovial fluid, cerebrospinal fluid (CSF), and urine. Meta-analyses show the strengths and weaknesses of these methods. Overall, assays for skin and synovial fluid (68% and 73%, respectively) have high sensitivity and uniformity. The low test sensitivity of CSF (18%) and plasma (29%), variable sensitivities among CSF and urine assays, and persistence of Borrelia burgdorferi DNA in urine and synovial fluid even with therapy and convalescence make these unsuitable for primary diagnosis. Molecular assays for Lyme disease are best used with other diagnostic methods and only in situations in which the clinical probability of Lyme disease is high.

Skin biopsies of 36 patients with erythema migrans and acrodermatitis chronica atrophicans (ACA) before therapy and those of 8 patients after therapy were examined for Borrelia burgdorferi DNA by PCR. Skin biopsies of 27 patients with dermatological diseases other than Lyme borreliosis and those of 10 healthy persons were examined as controls. Two different primer sets targeting 23S rRNA (PCR I) and 66-kDa protein (PCR II) genes were used. PCR was performed with freshly frozen tissue (FFT) and paraffin-embedded tissue (PET). For FFT specimens of erythema migrans, 73% were positive by PCR I, 79% were positive by PCR II, and 88% were positive by combining PCR I and II. For PET specimens, PCR was less sensitive (PCR I, 44%; PCR II, 52%). For FFT specimens of ACA, PCR I was positive for two of five patients and PCR II was positive for four of five patients. B. burgdorferi was cultured from 79% of the erythema migrans specimens but not from any of the ACA lesions. Elevated B. burgdorferi antibodies were detected in sera of 74% of erythema migrans patients and 100% of ACA patients. All urine samples were negative by PCR II, whereas PCR I was positive for 27%. However, hybridization of these amplicons was negative. Sequencing of three amplicons identified nonborrelial DNA. In conclusion, urine PCR is not suitable for the diagnosis of skin borreliosis. A combination of two different primer sets achieves high sensitivity with skin biopsies. In early erythema migrans infection, culture and PCR are more sensitive than serology.

A nested PCR (nested flagellin PCR) carrying an internal E. coli DNA control was established and compared with an in-vitro culture method for the detection of Borrelia burgdorferi in urine specimens of dogs. The predicted specific amplicon of the flagellin gene fla was generated from all cultured strains of B. burgdorferi tested (comprising three European genospecies). In contrast, all 13 strains of seven other flagellated bacterial species were negative. The PCR detection limit yielded 20 cells of B. burgdorferi per ml of double-distilled water and approx. 250 bacteria per ml of dog urine. Using the bacterial culture method, urine specimens collected from 216 dogs in Germany were all diagnosed negative for spirochetes by in-vitro culture and dark-field microscopy. In contrast, DNA of B. burgdorferi was detected in 32 specimens (14.8%) by PCR. 31 urine specimens (14.4%) showed inhibitory activity in the PCR assay. However, 94 (44%) were inhibitory in the culture assay. The majority of the PCR-positive dogs exhibited major clinical symptoms which have not been reported in the course of B. burgdorferi infection previously, e.g. cystitis (14/32 dogs) or prostatitis (5/32 dogs). Our results indicate that the analysis of urine specimens by the nested flagellin PCR is a highly valuable procedure for the diagnosis of B. burgdorferi infections in dogs.

Borrelia burgdorferi specific DNA has been detected by polymerase chain reaction (PCR) in different specimens of patients with Lyme disease (LD). The aim of the present study is to evaluate PCR-diagnostic of urine specimens regarding rheumatologic diagnosis of Lyme disease. Urine specimens of 77 patients (LD, n = 34; undifferentiated arthritis (UA), n = 25; arthralgia/myalgia (AM), n = 18) and 15 controls were investigated. Flagellin gene (60 specimens) or OspA-plasmid (32 specimens) were used as targets. Sensitivity of the flagellin-nested-PCR was 27%, by OspA-nested-PCR only one positive PCR result was found. Despite of low sensitivity PCR enabled the correct diagnosis of LD in two patients classified as UA. Therefore, PCR can give valuable hints in single cases if LD is clinically suspected.

The clinical manifestations of human Lyme disease present with a spectrum of tissue or organ involvement and severity of symptoms. The murine model of Lyme disease has proved to be an accurate reflection of many of the human symptoms of disease and has been particularly useful for studying development of subacute arthritis and tendonitis. Direct tissue invasion by Borrelia burgdorferi and persistence of high levels of spirochetes in tissues are important components of arthritis development. The outer-surface lipoproteins contain a biologically active lipid-modified moiety with potent ability to stimulate inflammatory cytokine production and other inflammatory mediators such as nitric oxide. Localized inflammation stimulated by these lipoproteins may be the trigger for neutrophil infiltration, synovial proliferation, and other events associated with this arthritis. Invasion of maternal uterine tissue, but not direct invasion of fetal tissue, is associated with low levels of pregnancy loss in mice infected during gestation, consistent with the detrimental effect of inflammatory cytokines on pregnancy.

The presence of Borrelia burgdorferi DNA was established by PCR from urine samples of 97 patients clinically diagnosed as presenting with symptoms of chronic Lyme disease. All patients had shown erythema chronica migrans following a deer tick bite. Most of the patients had been antibiotic-treated for extended periods of time. We used three sets of primer pairs with DNA sequences for the gene coding of outer surface protein A (OspA) and of a genomic sequence of B. burgdorferi to study samples of physician-referred patients from the mideastern USA. Controls from 62 healthy volunteers of the same geographic areas were routinely carried through the procedures in parallel with patients' samples. Of the 97 patients, 72 (74.2%) were found with positive PCR and the rest with negative PCR. The 62 healthy volunteers were PCR negative. It is proposed that a sizeable group of patients diagnosed on clinical grounds as having chronic Lyme disease may still excrete Borrelia DNA, and may do so in spite of intensive antibiotic treatment.

To simplify detection procedures of DNA fragments resulting from PCR, we developed a colorimetric microplate hybridization assay. This format was used for the identification of Borrelia burgdorferi sensu lato, the causal agent of Lyme disease. The system relied on the use of a specific capture probe covalently linked to polystyrene plates and a specific polybiotinylated detection probe. DNA fragments, resulting from PCR and sandwiched between these two probes, were detected by enzymatic color development. The new detection format outperformed agarose gel electrophoresis of PCR products in sensitivity and specificity Moreover, in view of its rapidity and simplicity, the system proved appropriate for the routine diagnostic analysis of clinical specimens from Lyme disease patients. The proposed detection format can be adapted easily to other DNA targets and is suitable for automation.

It is difficult in some cases to identify an infection caused by Borrelia burgdorferi and to monitor the effect of therapy. Seropositivity will persist even after successful treatment and therefore may suggest ongoing infection. For direct detection of B. burgdorferi DNA in human urine samples, the polymerase chain reaction (PCR) was evaluated. A published primer system was selected, which amplifies a 259 bp fragment from the gene encoding the 23S rRNA. The lower detection limit of the primer system was 10 fg of extracted B. burgdorferi DNA. Several methods for the pretreatment of urine samples were tested. Of these, the Geneclean kit (Bio 101, USA) showed the best results. A total of 114 urine samples from 74 patients belonging to three clinical groups was investigated: (i) 51 samples from 26 patients with active Lyme disease, (ii) 36 samples from 27 patients with previous infection but no symptoms at the time the urine was collected, and (iii) 27 samples from 21 seronegative control patients without Lyme disease. B. burgdorferi DNA was detected in 25 urine samples of 17 patients with active disease, whereas 26 samples from this group of patients were negative. Only one asymptomatic case with previous infection showed a positive result, and the urine samples of the patients without Lyme disease were uniformly negative. Two of four patients from whom samples before and directly after onset of therapy were available converted from negative to positive PCR results after initiation of therapy, accompanied by the symptoms of a Jarisch-Herxheimer reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Since the possibility of asymptomatic infection with Borrelia burgdorferi has been suggested by a positive serology found in healthy subjects, we hypothesized that these subjects might excrete borrelial DNA sequences in urine as happens in patients with Lyme borreliosis. We found borrelial sequences by nested PCR in the urine samples from 3 of 13 healthy B. burgdorferi antibody-positive adults but not in urine samples from 79 antibody-negative healthy controls. After therapy with doxycycline, the urine samples were repeatedly negative for B. burgdorferi DNA. We conclude that urinary excretion of borrelial DNA sequences may occur in seropositive healthy subjects during asymptomatic infection. Demonstration of such sequences in urine must be interpreted cautiously and may not necessarily prove a borrelial cause of disease.

The laboratory diagnosis of Lyme disease has been problematic because of the lack of sufficient sensitivity and specificity of the many tests used as well as the lack of interlaboratory standardization in the performance of these tests. Although helpful, the laboratory tests can only serve to support the clinical findings in making the diagnosis of active Lyme disease, the exception being a positive culture of Borrelia Burgdorferi.

Diagnosis of neuroborreliosis is often difficult since history and clinical presentation may be non-specific and serological tests may initially be negative. We therefore tested the polymerase chain reaction (PCR) for the detection of borrelial sequences in CSF and urine samples of consecutive children with neuroborreliosis seen in a single summer season. Four of eight children were negative in serum for antibodies to Borrelia burgdorferi. Two of eight children were PCR-positive in CSF and one other child was positive in urine. In two out of four children PCR was the only laboratory test confirming the clinical diagnosis. All children recovered after treatment with third generation cephalosporins. When seven of eight children were re-examined 6 months later all were healthy and antibodies to B. burgdorferi were detected in their serum. PCR may assist the paediatrician in establishing a diagnosis of neuroborreliosis; however, a negative result does not rule out neuroborreliosis. PCR is an adjunct, but no substitute for clinical judgement and serology.

A polymerase chain reaction (PCR) was developed for use in the identification of a 248-bp fragment of the Borrelia burgdorferi flagellin gene in urine and cerebrospinal fluid (CSF) from patients with Lyme neuroborreliosis. The specificities of the PCR products were confirmed by DNA-DNA hybridization with an internal probe. The assay had a detection limit of 10 in vitro-cultivated B. burgdorferi. The PCR assay seemed to be species wide as well as species specific, since DNA from all 21 B. burgdorferi isolates from humans tested but not from Borrelia hermsii or Treponema pallidum could be amplified. We tested 10 consecutively diagnosed patients with untreated neuroborreliosis. There was lymphocytic pleocytosis and intrathecal B. burgdorferi-specific antibody synthesis in the CSF of all patients. Urine and CSF samples were investigated by PCR before, during, and up to 8.5 months after therapy. B. burgdorferi DNA was detected in urine samples from nine patients; five patients, including two patients with chronic neuroborreliosis, were PCR positive prior to treatment, whereas urine samples from the remaining four patients obtained 3 to 6 days after the onset of therapy became PCR positive. All urine samples obtained greater than 4 weeks after therapy were negative by PCR. PCR of CSF was less sensitive, and samples from only four patients, including one with chronic neuroborreliosis, were positive. We conclude that urine is a more suitable sample source than CSF for use in B. burgdorferi DNA detection by PCR. Normalization of inflammatory CSF changes and the negative PCR results during follow-up even in patients with chronic neuroborreliosis do not point to a persistent infection. The future role of PCR as a diagnostic tool for Lyme neuroborreliosis is still uncertain.

Current diagnostic tests for Lyme disease (LD) are dependent upon the host serologic response and are insensitive early in infection and, possibly, following antibiotic therapy. We cloned a library of Borrelia burgdorferi 297 DNA and studied one clone, Ly-1, for its potential in diagnostic and pathogenic studies. Using pulsed-field electrophoresis, we demonstrated that Ly-1 is of chromosomal origin and estimated that the B. burgdorferi chromosome is approximately 1,100 kb in size. The 3.7-kb Ly-1 clone hybridizes with geographically diverse strains of B. burgdorferi. No cross hybridization occurs with DNA from human cells, Escherichia coli, Staphylococcus aureus, Clostridium difficile, or the closely related B. hermsii. We used a dot blot assay to detect 100 pg of B. burgdorferi DNA. We partially determined the nucleotide sequence of Ly-1 and used it to select and synthesize oligonucleotides for use in the polymerase chain reaction (PCR). Two different primer pairs were found to amplify DNA from nine geographically diverse isolates. We could detect 10 fg (less than 10 molecules) of B. burgdorferi or less than five spirochetes added to human urine. Finally, we were able to use the PCR to detect B. burgdorferi DNA in the urine of four of eight patients with suspected active LD (three with arthritis and one with neurologic manifestations), all of whom responded to antibiotic treatment. In contrast, those patients who were PCR negative either had inactive disease or had been appropriately treated and did not respond to additional antibiotics, and all four control urine specimens were PCR negative. We conclude that B. burgdorferi DNA can be sensitively detected by the PCR with the primers and methods we describe and that the urinary tract is a site of persistent infection in some cases of human LD, an observation of potential diagnostic and pathogenic importance.

Segments of the ospA gene of Borrelia burgdorferi were amplified by the polymerase chain reaction (PCR). Oligonucleotide primers used in the reaction flank a 309-base-pair segment within the ospA gene. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis or dot hybridization with a 32P-labeled probe. This segment was amplified in all strains of B. burgdorferi tested, but it was not detected in other bacterial species. An additional primer pair which has a broad specificity for conserved 16S rRNA sequences that are present in eubacteria amplified a 215-base-pair fragment in all organisms tested. The sensitivity of PCR for the detection of B. burgdorferi in clinical samples was evaluated by seeding blood and urine specimens with B. burgdorferi and subjecting them to amplification. We were able to detect 10 organisms per ml of blood or urine, using PCR with dot hybridization detection. DNA extraction is not required for sample preparation. Blood and urine specimens were obtained from canines with clinical and serologic evidence of Lyme disease and subjected to PCR analysis. Of 17 clinical specimens from 15 animals, one blood specimen showed reactivity in the PCR.